Kiyoshi Yasukawa

Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin.

When stimulated with antigen, B cells are influenced by T cells to proliferate and differentiate into antibody-forming cells. Since it was reported1,2 that soluble factors could replace certain functions of helper T cells in the antibody response, several different kinds of lymphokines and monokines have been reported in B-cell growth and differentiation3,4. Among these, human B-cell differentiation factor (BCDF or BSF-2) has been shown to induce the final maturation of B cells into immunoglobulin-secreting cells5–8. BSF-2 was purified to homogeneity9 and its partial NH2-terminal amino-acid sequence was determined10. These studies indicated that BSF-2 is functionally and structurally unlike other known proteins. Here, we report the molecular cloning, structural analysis and functional expression of the cDNA encoding human BSF-2. The primary sequence of BSF-2 deduced from the cDNA reveals that BSF-2 is a novel interleukin consisting of 184 amino acids.

Interleukin-6 triggers the association of its receptor with a possible signal transducer, gp130.

Interleukin-6 mediates pleiotropic functions in various types of cells through its specific receptor (IL-6-R), the cDNA of which has already been cloned. We report here that an 80 kd single polypeptide chain (IL-6-R) is involved in IL-6 binding and that IL-6 triggers the association of this receptor with a non-ligand-binding membrane glycoprotein, gp130. The association takes place at 37 degrees C within 5 min and is stable for at least 40 min in the presence of IL-6, but does not occur at 0 degree C. Human IL-6-R can associate with a murine gp130 homolog and is functional in murine cells. Mutant IL-6-R lacking the intracytoplasmic portion is functional, suggesting that the two polypeptide chains interact to involve their extracellular portion. In fact, a soluble IL-6-R lacking the transmembrane and intracytoplasmic domains can associate with gp130 in the presence of IL-6 and mediate its function. These findings indicate that the complex of IL-6 and IL-6-R can interact with a non-ligand-binding membrane glycoprotein, gp130, extracellularly and can provide the IL-6 signal.

CNTF and LIF act on neuronal cells via shared signaling pathways that involve the IL-6 signal transducing receptor component gp130

Ciliary neurotrophic factor (CNTF) has a variety of actions within the nervous system. While some of the actions of leukemia inhibitory factor (LIF) on neurons resemble those of CNTF, LIF also has broad actions outside of the nervous system that in many cases mimic those of interleukin-6 (IL-6). Comparison of the tyrosine phosphorylations and gene activations induced by CNTF and LIF in neuron cell lines reveals that they are indistinguishable and also very similar to signaling events that characterize LIF and IL-6 responses in hematopoietic cells. We provide a basis for the overlapping actions of these three factors by demonstrating that the shared CNTF and LIF signaling pathways involve the IL-6 signal transducing receptor component gp130. Thus, the receptor system for CNTF is surprisingly unlike those used by the nerve growth factor family of neurotrophic factors, but is instead related to those used by a subclass of hematopoietic cytokines.

Structure and expression of human B cell stimulatory factor-2 (BSF-2/IL-6) gene

The chromosomal DNA segment of human B cell stimulatory factor‐2 (BSF‐2/IL‐6) was isolated and characterized by nucleotide sequence analysis. The human BSF‐2/IL‐6 gene consists of five exons and four introns and its organization shows a distinctive similarity to granulocyte colony‐stimulating factor gene. The two genes have the same number of exons and introns and the size of each exon is strikingly similar. The BSF‐2/IL‐6 mRNA was found to be constitutively expressed in a human T cell leukemia virus‐1 transformed T cell line, TCL‐Na1, a bladder cell carcinoma line, T24, and an amnion derived cell line, FL. The BSF‐2/IL‐6 mRNA was also found to be inducible with interleukin‐1 beta in an astrocytoma line, U373 and a glioblastoma line, SK‐MG‐4. S1 mapping and primer extension analyses showed the presence of multiple initiation sites and the preferential utilization of a different initiation site for each individual tissue tested.

Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor

Here, we demonstrate a significant ex vivo expansion of human hematopoietic stem cells capable of repopulating in NOD/SCID mice. Using a combination of stem cell factor (SCF), Flk2/Flt3 ligand (FL), thrombopoietin (TPO), and a complex of IL-6 and soluble IL-6 receptor (IL-6/sIL-6R), we cultured cord blood CD34(+) cells for 7 days and transplanted these cells into NOD/SCID mice. Bone marrow engraftment was judged successful when recipient animals contained measurable numbers of human CD45(+) cells 10-12 weeks after transplantation. When cells were cultured with SCF+FL+TPO+IL-6/sIL-6R, 13 of 16 recipients were successfully engrafted, and CD45(+) cells represented 11.5% of bone marrow cells in engrafted recipients. Cells cultured with a subset of these factors were less efficiently engrafted, both as measured by frequency of successful transplantations and prevalence of CD45(+) cells. In animals receiving cells cultured with all 4 factors, human CD45(+) cells represented various lineages, including a large number of CD34(+) cells. The proportion of CD45(+) cells in recipient marrow was 10 times higher in animals receiving these cultured cells than in those receiving comparable numbers of fresh CD34(+) cells, and the expansion rate was estimated at 4.2-fold by a limiting dilution method. Addition of IL-3 to the cytokine combination abrogated the repopulating ability of the expanded cells. The present study may provide a novel culture method for the expansion of human transplantable hematopoietic stem cells suitable for clinical applications.

Maintenance of the pluripotential phenotype of embryonic stem cells through direct activation of gp130 signalling pathways

Propagation of the undifferentiated pluripotential phenotype of embryonic stem (ES) cells is dependent on the cytokine differentiation inhibiting activity/leukemia inhibitory factor (DIA/LIF). The DIA/LIF receptor complex is a heterodimer of DIA/LIF receptor (DIA/LIF-R) and gp130. The latter is also a component of the interleukin-6 (IL-6) receptor complex. We report that a combination of IL-6 and soluble IL-6 receptor (sIL-6R), which can induce homodimerisation of gp130 and activation of signalling processes, sustains self-renewal of pluripotential ES cells. Our findings indicate that the IL-6/sIL-6R complex acts on ES cells through gp130 alone, bypassing DIA/LIF-R, and therefore implicate gp130 as the key component in the signalling pathway responsible for stem cell renewal.

Structure-function analysis of human IL-6 receptor : dissociation of amino acid residues required for IL-6-binding and for IL-6 signal transduction through gp130

Here, we report the analysis of the structure‐function relationship of the extracellular region of human interleukin 6 receptor (IL‐6R). Upon binding of IL‐6, IL‐6R becomes associated extracellularly with a non‐IL‐6‐binding but signal transducing molecule, gp130, and the IL‐6 signal is generated. In this region, the cytokine receptor family domain, but not the immunoglobulin‐like domain, was responsible both for IL‐6 binding and for signal transduction through gp130. Because a soluble, extracellular portion of IL‐6R (sIL‐6R) could bind IL‐6 and mediate IL‐6 functions through gp130, amino acid substitutions were introduced into sIL‐6R by site‐directed mutagenesis. The results, together with the previously proposed tertiary structure model, suggested that the amino acid residues critical for IL‐6 binding have a tendency to be distributed to the hinge region between the two ‘barrel’‐like fibronectin type III modules and to the same side of these two ‘barrels’. Amino acid residues, of which substitutions barely affected the IL‐6‐binding but did abolish the IL‐6 signalling capability of sIL‐6R, were identified and found to be located mainly in the membrane proximal half of the second barrel. sIL‐6R mutants carrying such substitutions lacked the capacity to associate with gp130 in the presence of IL‐6.

Differential shedding of the two subunits of the interleukin‐6 receptor

cDNAs coding for the two receptor subunits of the interleukin‐6 receptor have been stably expressed in Madine Darby canine kidney (MDCK) cells. The fate of the IL‐6 binding protein (IL‐6R) and of the signal transducing protein gp130 was studied independently. Both proteins were proteolytically cleaved from cells metabolically labeled with [35S]methionine/cysteine leading to the release of soluble receptor proteins of 55 kDa and 100 kDa, respectively. In contrast to the shedding of the IL‐6R gp 130 was inefficiently released from the cells and the process was not significantly stimulated by the phorbolester PMA. In addition we show that the soluble forms of the IL‐6R and gp 130 released by transfected cells can form a ternary complexe with interleukin‐6 indicating that such complexes also may occur in vivo. gp 130; Interleukin‐6; Interleukin‐6‐receptor; Protein kinase C; Shedding

Combination of Interleukin-6 and Soluble Interleukin-6 Receptors Induces Differentiation and Activation of JAK-STAT and MAP Kinase Pathways in MG-63 Human Osteoblastic Cells

Studies on the role of interleukin‐6 (IL‐6) in bone metabolism have been accumulating. However, its effects on osteoblasts are still unclear because the results are conflicting depending on the study models employed. We reasoned that these conflicting data are due to variable expression levels of membrane‐bound IL‐6 receptors (IL‐6Rs). In the present study, we found that IL‐6 in combination with soluble IL‐6R (sIL‐6R) consistently caused a marked elevation of alkaline phosphatase and a decrease in proliferation in the human osteoblastic cell line MG‐63, which expressed no detectable membrane‐bound IL‐6R and failed to respond to IL‐6. These effects of IL‐6/sIL‐6R were blocked by neutralizing antibodies to the IL‐6 signal transducer gp130, suggesting an involvement of IL‐6 signaling in the elicitation of the effects of IL‐6/sIL‐6R. Upon stimulation with IL‐6/sIL‐6R, the gp130, cytoplasmic Janus kinases JAK1 and JAK2 were tyrosine phosphorylated. Moreover, signal transducers and activators of transcription STAT1 and STAT3 were also tyrosine phosphorylated, translocated to the nucleus, and bound to the putative STAT‐binding DNA elements. In addition, mitogen‐activated protein (MAP) kinase was also activated in response to IL‐6/sIL‐6R. These data demonstrate that sIL‐6R may enhance the responsiveness of MG‐63 cells to IL‐6. Thus, IL‐6 in collaboration with sIL‐6R may modulate differentiation and proliferation of osteoblastic cells, presumably by activating two distinct signaling pathways of JAK‐STAT and MAP kinase.

Coadministration of interleukin-6 (IL-6) and soluble IL-6 receptor delays progression of wobbler mouse motor neuron disease

Interleukin-6 (IL-6), a multipotential cytokine, initiates signal transduction pathways similar to those of ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF). These molecules share the signal transducing receptor component, gp130. IL-6 triggers homodimerization of gp130, whereas CNTF and LIF induce heterodimerization of gp130 and LIF receptor. Although CNTF or LIF treatment attenuates motor deficits in wobbler mouse motor neuron disease (MND), neuroprotective effects of IL-6 on this animal have not yet been clarified. Here we studied whether simultaneous treatment with IL-6 and soluble IL-6 receptor (sIL-6R) can ameliorate symptomatic and neuropathological changes in wobbler mouse MND. After clinical diagnosis at postnatal age 3-4 weeks, wobbler mice received subcutaneous injection with human recombinant IL-6 (1.0 mg/kg), human sIL-6R (0.5 mg/kg), IL-6 + sIL-6R or vehicle, daily for 4 weeks in a blind fashion. Compared to vehicle, coadministration with IL-6 and sIL-6R potentiated grip strength, attenuated muscle contractures in the forelimbs, reduced denervation muscle atrophy and prevented degeneration of spinal motor neurons. Single administration with IL-6 or sIL-6R did not retard the symptomatic and neuropathological progression, although IL-6-treated mice did not raise anti-IL-6 antibodies. Treatment with IL-6 + sIL-6R, but not with IL-6 or sIL-6R alone delayed progression of wobbler mouse MND. Our results indicate that the neuroprotective mechanism for IL-6/sIL-6R on wobbler mouse MND differs from that of CNTF or LIF alone. We hypothesize that IL-6/sIL-6R complex may function on motor neurons through activation and homodimerization of gp130.